Volume 24 Number 8| August 2018| Dermatology Online Journal || Case Presentation 24(8): 10 Enzyme-linked immunosorbent assay as a helpful diagnostic tool for pemphigus erythematosus with equivocal histologic and immunofluorescent findings Anand Desai1 BS, Jessica Harris2 MD, Kiran Motaparthi2 MD Affiliations: 1Department of Dermatology, University of Central Florida College of Medicine, Orlando, Florida, USA, 2Department of Dermatology, University of Florida College of Medicine, Gainesville, Florida Corresponding Author: Kiran Motaparthi MD, Department of Dermatology, University of Florida College of Medicine, 4037 NW 86th Terrace, 4th floor, Springhill Room 4119, Tel: 352-594-1930, Fax: 352-594-1926, Email: [email protected] commonly utilized diagnostic confirmatory method Abstract [3, 4]. However, recent evidence identifies ELISA as Enzyme-linked immunosorbent assay is a more accurate than DIF and histopathology for the sensitive and specific method for the detection diagnosis of pemphigus vulgaris (PV) and PF. Less of circulating autoantibodies in pemphigus evidence supports the utility of ELISA for the vulgaris and foliaceus. Herein, pemphigus diagnosis of uncommon variants of pemphigus, such erythematosus with equivocal immunofluo- as pemphigus erythematosus (PE). rescence and non-diagnostic histology, but confirmed by enzyme-linked immunosorbent assay, is described. As a non-invasive, sensitive, Case Synopsis and specific assay with additional utility for A 65-year-old woman with a history of rheumatoid monitoring disease activity, this case adds to arthritis presented for evaluation of a chronic growing evidence supporting ELISA as the blistering eruption of 6 months duration associated diagnostic method of choice for common and with pruritus. Multiple scattered ruptured bullae and less common variants of pemphigus. erosions with crust and hyperpigmentation were present on her chest and upper back (Figure 1A). Mucosal involvement was absent. Histopathology Keywords: pemphigus foliaceus, autoimmune blistering failed to identify a discrete intraepidermal split, but disorder, immunofluorescence, enzyme-linked eosinophilic spongiosis was demonstrated (Figure immunosorbent assay 1B). DIF of perilesional skin revealed granular IgM, IgG, C3, and C5b-9 deposition at the Introduction dermoepidermal junction (Figures 2A-2D, respectively). Weak, focal cell surface IgG reactivity Pemphigus foliaceus (PF) is an autoimmune disease (Figure 2B) was considered equivocal. Owing to the characterized by superficial blisters. It is caused by keratinocyte nuclear immunofluorescence (in vivo immunoglobulin (Ig) G autoantibodies that target antinuclear antibody [ANA], Figure 2B), this lupus desmoglein (DSG) 1 and is the second most band was suggestive of autoimmune connective prevalent subtype of pemphigus [1, 2]. tissue disease (AICTD) such as systemic lupus Immunofluorescence, histology, and more recently, erythematosus (SLE). enzyme-linked immunosorbent assay (ELISA) support the clinical diagnosis of pemphigus. Given the association of eosinophilic spongiosis with Selecting the best test for diagnosis may pose a clinical findings of an autoimmune bullous disorder, challenge. Direct immunofluorescence (DIF), in ELISAs for autoantibodies against bullous tandem with routine histology, is the most pemphigoid antigen (BPAG) 1 and 2 and DSG 1 and 3 - 1 - Volume 24 Number 8| August 2018| Dermatology Online Journal || Case Presentation 24(8): 10 /Table Figure 1. A) Healed superficial bullae, crusted erosions, and post-inflammatory hyperpigmentation on the upper back. B) Mild spongiosis with rare eosinophils present in the basilar epidermis. H&E, 200×. were evaluated. Anti-DSG1 was 120U (reference initially sufficient, but subsequent flaring required range: negative <14, positive >20), diagnostic of PF; addition of mycophenolate mofetil, which produced the other 3 serologies were negative. Rheumatoid remission. factor level was 127 IU/mL and IgG for cyclic citrulline peptide IgG was measured at 166U. Treatment with class II topical corticosteroids over 6 weeks was Figure 3. A) Localized, thin plaques with fine scale with a Figure 2. A) Granular IgM deposition at the dermoepidermal seborrheic distribution in a patient with localized pemphigus junction. Anti-IgM, 400×. B) Granular IgG deposition and the foliaceus. B) Prominent acantholysis involves the granular layer basement membrane along with in vivo antinuclear antibody, of the follicular infundibulum; rare dyskeratosis (arrow) is also demonstrated by keratinocyte nuclear staining (blue arrow). Red present. H&E, 200×. C) Follicular spongiosis and acantholysis arrow indicates weak focal (equivocal) cell surface IgG deposition. along with rare dermal eosinophils (arrow). H&E, 200×. d) Focal Anti-IgG, 400×. C) Prominent granular C3 deposition at the intercellular IgG deposition limited to the superficial (granular) dermoepidermal junction (Anti-C3, 400×. D) C5b-9 junctional epidermis, without junctional immunoreactant deposition. Anti- deposition was also identified. Anti-C5b-9, 400×. IgG, 400×. - 2 - Volume 24 Number 8| August 2018| Dermatology Online Journal || Case Presentation 24(8): 10 Table 1. Sensitivity and specificity of DIF, IIF, and ELISA for the malar regions (Figure 3). By contrast, classic PE, as an diagnosis of PV and PF. immunopathologic variant represented by the case presented herein (Figures 1 and 2), is characterized PV PF by overlapping features of SLE and PF, including Sensitivity Specificity Sensitivity Specificity deposition of multiple immunoreactants in a DIF [8, Approaches Approaches 36% 36% 9] 100% 100% granular pattern (analogous to that seen in lupus band), keratinocyte nuclear fluorescence (in vivo IIF [9] 81-95% 92% 81-95% 81-95% ANA), and IgG on keratinocyte cell surfaces. In vivo ELISA 97% 98% 96% 99% ANA is also demonstrable in dermatomyositis, [1] DIF: direct immunofluorescence; IIF: indirect immunofluorescence; subacute cutaneous lupus erythematosus, and ELISA: enzyme-linked immunosorbent assay; PV: pemphigus vulgaris; mixed connective tissue disease. Serologic PF: pemphigus foliaceus evaluation may also demonstrate overlapping features of SLE, including ANA with significant titers Case Discussion in 40% of patients [5]. PE, also known as Senear-Usher syndrome, remains a controversial nosologic entity with variable criteria Recent studies have demonstrated that ELISA is a for diagnosis. When considered as a clinical subtype highly sensitive and specific method for diagnosing of PF, it has been described as a localized or early pemphigus. ELISA for anti-DSG1 boasts a sensitivity variant, with a predilection for the seborrheic and/or of 96% and a specificity of 99% [6]. The sensitivity Table 2. Cases of PE with diagnostic support by ELISA [18,19,20]. Associated diseases and Age and Clinical laboratory Author gender findings Histology DIF ELISA findings Gomi et al. Erosions Superficial CS: IgG Anti-DSG1: 30; F SLE [20] localized to face acantholysis DEJ: IgG, IgM, C3 170 Generalized Anti-DSG1: RA Karlhofer et erosions on the Superficial CS: IgG, C3 199 43; F ANA 1:160 al. [18] face, trunk, and acantholysis DEJ: granular C3 Anti-BPAG1: RF: 56.5 IU/mL extremities 29 Generalized Anti-DSG1: SLE Karlhofer et erosions on the Superficial CS: IgG, C3 177 ANA 1:320 22; F al. [18] face, trunk, and acantholysis DEJ: granular C3 Anti-BPAG1: Anti-dsDNA: 48.5 extremities 28 IU/mL Erosions Anti-DSG1: 32 Karlhofer et Superficial CS: IgG, C3 34; F localized to the Anti-BPAG1: al. [18] acantholysis DEJ: granular C3 chest 16 43 Akman et al. Superficial CS: IgG Anti-DSG1: (mean); 3 NS ANA negative (n=5) [19] acantholysis DEJ: linear C3, IgG 135 (mean) M, 2 F Eosinophilic CS: weak or equivocal Superficial RA spongiosis IgG bullae localized Anti-DSG1: ANA Desai et al. 65; F without DEJ: granular IgM, to chest and 120 RF 127 IU/mL intraepidermal IgG, C3, C5b-9 back CCP: 166 U split In vivo ANA PE: pemphigus erythematosus; ELISA: enzyme-linked immunosorbent assay; DIF: direct immunofluorescence; M: male; F: female; CS: cell surface; DEJ: dermoepidermal junction; IgG: immunoglobulin G; DSG: desmoglein; BPAG1: bullous pemphigoid antigen 1; RA: rheumatoid arthritis; ANA: antinuclear antibody (normal < 1:40); RF: rheumatoid factor (normal 0-15 IU/mL); SLE: systemic lupus erythematosus; dsDNA: double-stranded deoxyribonucleic acid (normal 0-7 IU/mL); NS: not specified; CCP: cyclic citrulline peptide IgG (normal 0-19 U) - 3 - Volume 24 Number 8| August 2018| Dermatology Online Journal || Case Presentation 24(8): 10 and specificity of ELISA for anti-DSG3 in PV have sensitive and specific diagnostic tests are been reported to be over 95% and 98%, respectively immunoblotting for envoplakin and/or periplakin [7]. DIF approaches 100% sensitivity but cannot (sensitivity 100% and specificity 82-91%) and IIF on definitively distinguish PF from PV as both transitional epithelium (rat bladder, sensitivity 67- demonstrate bound intercellular IgG and 95%, specificity 100%), respectively [16, 17]. complement [8, 9]. Thus, ELISA is almost as sensitive In contrast to more common variants of pemphigus, as, and more specific than, DIF for PF and PV. the utility of ELISA has been only infrequently By examining the level of acantholysis, one can often described for PE [18-20]. Given the serologic and distinguish pemphigus vulgaris (PV) from PF. clinical overlap with AICTDs including SLE, and the However, up to 25% of biopsies of PV and PF reported co-presence of circulating autoantibodies